Conformational studies of biliproteins from the insects pieris brassicae and cerura vinula

Abstract

Chromophore conformation and protein secondary structure of biliproteins from the butterfly, Pieris brassicae, and the moth. Cerura vinula, have been investigated by absorption, circular dichroism and fluorescence spectroscopy. The chromophore of the P. brassicae protein, biliverdin ΙΧγ, has probably a cyclic-helical structure similar to that of free bile pigments of the biliverdin type. Though achiral by structure the chromophore displays strong optical activity in the native protein-bound state, but becomes inactive after urea denaturation of the protein. A minor biliprotein from P. brassicae shows absorption, circular dichroism and fluorescence spectra identical to the main biliprotein. In the biliprotein from Cerura vinula the structure of the pigment is still unknown. It has a semi-open conformation intermediate between that of the Pieris proteins and that of the phycobiliprotein. C-phycocyanin, and it retains optical activity after urea denaturation. The band widths and the size of the Stokes shifts of the fluorescence spectra indicate a high degree of conformational flexibility of the chromophores in the two Pieris pigments, and a decreased flexibility in the one from Cerura. In the biliproteins from both insects the polypeptides are low in α-helix content compared to that of phycobiliproteins. From these and earlier data, insect and algal biliproteins seem to be related only distantly if at all. but there exist also considerable differences among insect biliproteins from different species.

The bilin‐binding protein of Pieris brassicae

“…The visible absorption band had a composite structure with close resemblance to the natural protein complex and an identical A675/A383 ratio of 0.56. The absorbance values for the bound ligand at 383 nm and the protein component at 280nm showed a ratio of 0.75 compared to a value of 1.0 earlier determined for the natural holo-BBP (Scheer and Kayser, 1988). In similar reconstitution experiments with biliverdin IXa and protoporphyrin IX, complex formation could not be detected (data not shown).…”

“…The absorption coefficients used were E~~~ of 54500 cm-' M-' (in methanol) for biliverdin 1x1, (Heirwegh et al, 1991), E~~~ of 64200, E~~~ of 64200, E~~~ of 36300 cm-' M-' for holo-BBP (Scheer and Kayser, 1988) and &280 of 50555 cm-' M-' for recombinant apo-BBP as determined by the ratio A,,,/A,, according to Scopes (1974).…”

“…Holo-BBP prepared from P. brassicae has an absorption spectrum with a maximum at 383 nm and a broadened, complex absorption band around 675 nm, which is characteristic of the bilatriene chromophore (Huber et al, 1987b;Scheer and Kayser, 1988). The absorption spectrum (Fig.…”

BILIPROTEINS FROM THE BUTTERFLY Pieris brassicae STUDIED BY TIME‐RESOLVED FLUORESCENCE AND COHERENT ANTI‐STOKES RAMAN SPECTROSCOPY

“…Absorption and stationary emission spectra were recorded before and after each of the described measurements to check for photoinduced deterioration; the absorption spectra were identical to those shown by Scheer and Kayser (1988).…”

“…In the latter case the chromophore, namely biliverdin IXy (Rudiger, 1971), is not covalently linked to the apoprotein. From absorption, emission and circular dichroism studies Scheer and Kayser (1988) concluded that the chromophore should adopt a cyclic helical structure in the protein-bound state, whereas in phycobiliproteins from e.g. blue-green algae, the chromophore is held in an extended conformation by specific chromophore-protein interaction (see Fig.…”